Gallbladder epithelial cells and cholangiocytes are exposed to high concentrations of cholesterol because the major pathway of cholesterol excretion from the body is through bile. Oxidized forms of cholesterol, called oxysterols, are also found in bile. The effects of oxysterols on the biliary system are not understood. Cholesterol handling biliary epithelial cells is therefore an important are of investigation, as an understanding of the mechanisms involved in cholesterol transport and of the effects of oxysterols at the cellular and molecular level has potential implications for therapy of cholesterol gallstone disease, cholesterolosis of the gallbladder, and inflammatory states affecting the biliary system such as cholecystitis and cholangitis. An understanding of biliary epithelial cell cholesterol transport may also provide insights into cholesterol handling by other polarized epithelial cells such as villous enterocytes. We propose to use gallbladder epithelial cells, cultured in a polarized fashion, to study cholesterol transport, with a focus on the interactive roles of the cholesterol transporter ABCA1 and the HDL receptor, SR-B1. Key questions to be addressed are the polarity of expression of these molecules and their functional interactions. We hypothesize that ABCA1 and SR-B1 are expressed on opposing sides of polarized gallbladder epithelial cells. Cholesterol influx is mediated by SR-B1from the apical surface, and cholesterol efflux from the basolateral surface by ABCA1. This mechanism allows cholesterol concentrations in bile to be regulated, and a set-point for cholesterol concentration maintained based on the relative activities of these proteins. We postulate a key role for apolipoprotein A1 in this process. We also postulate that oxysterosis modulate ABCA1 activity via transport mechanisms involving the nuclear hormone receptors LXR alpha and RXR. The Specific Aims are to: 1. Demonstrate the functional expression of ABCA1activity via transcriptional mechanisms involving the nuclear hormone receptors LXR alpha and RXR. 2. Determine the mechanisms of apolipoprotein A1-mediated cholesterol efflux in polarized gallbladder epithelial cells. 3. Compare and contrast the mechanisms of activation of ABCA1 via the cAMP responsive and the LXRalpha/RXR- responsive pathways.